The tumor suppressor Rb is thought to control cell proliferation, survival and differentiation. We recently showed that differentiating Rb-deficient mouse myoblasts can fuse to form short myotubes that quickly collapse through a mechanism involving autophagy, and that autophagy inhibitors or hypoxia could rescue the defect leading to long, twitching myotubes. Here we determined the contribution of pRb relatives, p107 and p130, to this process. We show that chronic or acute inactivation of Rb plus p107 or p130 increased myoblast cell death and reduced myotube formation relative to Rb loss alone. Treatment with autophagy antagonists or hypoxia extended survival of double-knockout myotubes, which appeared indistinguishable from control fibers. In contrast, triple mutations in Rb, p107 and p130, led to substantial increase in myoblast death and to elongated bi-nuclear myocytes, which seem to derive from nuclear duplication, as opposed to cell fusion. Under hypoxia, some rare, abnormally thin triple knockout myotubes survived and twitched. Thus, mutation of p107 or p130 reduces survival of Rb-deficient myoblasts during differentiation but does not preclude myoblast fusion or necessitate myotube degeneration, whereas combined inactivation of the entire Rb family produces a distinct phenotype, with drastically impaired myoblast fusion and survival.

Signal transducer and activator of transcription 5 (STAT5) and nucleophosmin (NPM1) are critical regulators of multiple biological and pathological processes. Although a reciprocal regulatory relationship was established between STAT5A and a NPM-ALK fusion protein in T-cell lymphoma, no direct connection between STAT5 and wild-type NPM1 has been documented. Here we demonstrate a mutually regulatory relationship between STAT5 and NPM1. Induction of STAT5 phosphorylation at Y694 (P-STAT5) diminished NPM1 expression, whereas inhibition of STAT5 phosphorylation enhanced NPM1 expression. Conversely, NPM1 not only negatively regulated STAT5 phosphorylation but also preserved unphosphorylated STAT5 level. Mechanistically, we show that NPM1 downregulation by P-STAT5 is mediated by impairing the BRCA1-BARD1 ubiquitin ligase, which controls the stability of NPM1. In turn, decreased NPM1 levels led to suppression of p53 expression, resulting in enhanced cell survival. This study reveals a new STAT5 signaling pathway regulating p53 expression via NPM1 and uncovers new therapeutic targets for anticancer treatment in tumors driven by STAT5 signaling.

Multi-gene prognostic signatures derived from primary tumor biopsies can guide clinicians in designing an appropriate course of treatment. Identifying genes and pathways most essential to a signature performance may facilitate clinical application, provide insights into cancer progression, and uncover potentially new therapeutic targets. We previously developed a 17-gene prognostic signature (HTICS) for HER2+:ERα- breast cancer patients, using genes that are differentially expressed in tumor initiating cells (TICs) versus non-TICs from MMTV-Her2/neu mammary tumors. Here we probed the pathways and genes that underlie the prognostic power of HTICS.

Aerobic glycolysis is an important metabolic adaptation of cancer cells. However, there is growing evidence that reprogrammed mitochondria also play an important metabolic role in metastatic dissemination. Two constituents of the reprogrammed mitochondria of cancer cells are the intracellular tyrosine kinase Fer and its cancer- and sperm-specific variant, FerT. Here, we show that Fer and FerT control mitochondrial susceptibility to therapeutic and hypoxic stress in metastatic colon (SW620) and non-small cell lung cancer (NSCLC-H1299) cells. Fer- and FerT-deficient SW620 and H1299 cells (SW∆Fer/FerT and H∆Fer/FerT cells, respectively) become highly sensitive to metformin treatment and to hypoxia under glucose-restrictive conditions. Metformin impaired mitochondrial functioning that was accompanied by ATP deficiency and robust death in SW∆Fer/FerT and H∆Fer/FerT cells compared to the parental SW620 and H1299 cells. Notably, selective knockout of the fer gene without affecting FerT expression reduced sensitivity to metformin and hypoxia seen in SW∆Fer/FerT cells. Thus, Fer and FerT modulate the mitochondrial susceptibility of metastatic cancer cells to hypoxia and metformin. Targeting Fer/FerT may therefore provide a novel anticancer treatment by efficient, selective, and more versatile disruption of mitochondrial function in malignant cells.

CDH1 and PIK3CA are the two most frequently mutated genes in invasive lobular carcinoma (ILC) of the breast. Transcription profiling has identified molecular subtypes for ILC, one of which, immune-related (IR), is associated with gene expression linked to lymphocyte and macrophage infiltration. Here, we report that deletion of Cdh1, together with activation of Pik3ca in mammary epithelium of genetically modified mice, leads to formation of IR-ILC-like tumors with immune cell infiltration, as well as gene expression linked to T-regulatory (Treg) cell signaling and activation of targetable immune checkpoint pathways. Interestingly, these tumors show enhanced Rac1- and Yap-dependent transcription and signaling, as well as sensitivity to PI3K, Rac1, and Yap inhibitors in culture. Finally, high-dimensional immunophenotyping in control mouse mammary gland and IR-ILC tumors by mass cytometry shows dramatic alterations in myeloid and lymphoid populations associated with immune suppression and exhaustion, highlighting the potential for therapeutic intervention via immune checkpoint regulators.

Pediatric pineoblastomas (PBs) are rare and aggressive tumors of grade IV histology. Although some oncogenic drivers are characterized, including germline mutations in RB1 and DICER1, the role of epigenetic deregulation and cis-regulatory regions in PB pathogenesis and progression is largely unknown. Here, we generated genome-wide gene expression, chromatin accessibility, and H3K27ac profiles covering key time points of PB initiation and progression from pineal tissues of a mouse model of CCND1-driven PB. We identified PB-specific enhancers and super-enhancers, and found that in some cases, the accessible genome dynamics precede transcriptomic changes, a characteristic that is underexplored in tumor progression. During progression of PB, newly acquired open chromatin regions lacking H3K27ac signal become enriched for repressive state elements and harbor motifs of repressor transcription factors like HINFP, GLI2, and YY1. Copy number variant analysis identified deletion events specific to the tumorigenic stage, affecting, among others, the histone gene cluster and Gas1, the growth arrest specific gene. Gene set enrichment analysis and gene expression signatures positioned the model used here close to human PB samples, showing the potential of our findings for exploring new avenues in PB management and therapy. Overall, this study reports the first temporal and in vivo cis-regulatory, expression, and accessibility maps in PB.

Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic β-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity.

FLI1 is an oncogenic transcription factor that promotes diverse malignancies through mechanisms that are not fully understood. Herein, FLI1 is shown to regulate the expression of Ubiquitin Associated and SH3 Domain Containing A/B (UBASH3A/B) genes. UBASH3B and UBASH3A are found to act as an oncogene and tumor suppressor, respectively, and their combined effect determines erythroleukemia progression downstream of FLI1.

PTEN loss and PIK3CA activation both promote the accumulation of phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3). While these proteins also have distinct biochemical functions, beyond the regulation of PIP3, little is known about the consequences of these differences in vivo. Here, we directly compared cancer signalling in mammary tumors from MMTV-Cre:Ptenf/f and MMTV-Cre:Pik3ca(LSL-H1047R) mice. Using unsupervised hierarchical clustering we found that whereas MMTV-Cre:Pik3ca(LSL-H1047R)-derived tumors fall into two separate groups, designated squamous-likeEx and class14(Ex), MMTV-Cre:Ptenf/f tumors cluster as one group together with PIK3CA(H1047R) class14(Ex), exhibiting a 'luminal' expression profile. Gene Set Enrichment Analysis (GSEA) of Pten(Δ)ˆ† and PIK3CA(H1047R) class14(Ex) tumors revealed very similar profiles of signalling pathways as well as some interesting differences. Analysis of 18 signalling signatures revealed that PI3K signalling is significantly induced whereas EGFR signalling is significantly reduced in Pten(∆) versus PIK3CA(H1047R) tumors. Thus, Pten(∆) and PIK3CA(H1047R) tumors exhibit discernable differences that may impact tumorigenesis and response to therapy.

Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) encodes a tumor suppressor gene implicated in the growth of various tumor types including breast cancer. We previously demonstrated that recombinant adenovirus-mediated mda-7/IL-24 expression in the mammary glands of carcinogen-treated (methylnitrosourea, MNU) rats suppressed mammary tumor development. Since most MNU-induced tumors in rats contain activating mutations in Ha-ras, which arenot frequently detected in humans, we presently examined the effect of MDA-7/IL-24 on Her2/Neu-induced mammary tumors, in which the RAS pathway is induced. We generated tet-inducible MDA-7/IL-24 transgenic mice and crossed them with Her2/Neu transgenic mice. Triple compound transgenic mice treated with doxycycline exhibited a strong inhibition of tumor development, demonstrating tumor suppressor activity by MDA-7/IL-24 in immune-competent mice. MDA-7/IL-24 induction also inhibited growth of tumors generated following injection of Her2/Neu tumor cells isolated from triple compound transgenic mice that had not been treated with doxycycline, into the mammary fat pads of isogenic FVB mice. Despite initial growth suppression, tumors in triple compound transgenic mice lost mda-7/IL-24 expression and grew, albeit after longer latency, indicating that continuous presence of this cytokine within tumor microenvironment is crucial to sustain tumor inhibitory activity. Mechanistically, MDA-7/IL-24 exerted its tumor suppression effect on HER2+ breast cancer cells, at least in part, through PERP, a member of PMP-22 family with growth arrest and apoptosis-inducing capacity. Overall, our results establish mda-7/IL-24 as a suppressor of mammary tumor development and provide a rationale for using this cytokine in the prevention/treatment of human breast cancer.

The disruption of protein translation machinery is a common feature of cancer initiation and progression, and drugs that target protein translation offer new avenues for therapy. The translation initiation factor, eukaryotic initiation factor 4E (eIF4E), is induced in a number of cancer cell lines and is one such candidate for therapeutic intervention. Friend leukemia integration 1 (FLI1) is a potent oncogenic transcription factor that promotes various types of cancer by promoting several hallmarks of cancer progression. FLI1 has recently been implicated in protein translation through yet unknown mechanisms. This study identified a positive association between FLI1 expression and mitogen‑activated protein kinase (MAPK)‑interacting serine/threonine kinase1 (MKNK1), the immediate upstream regulator of the eIF4E initiation factor. The short hairpin RNA (shRNA)‑mediated silencing or overexpression of FLI1 in leukemic cell lines downregulated or upregulated MKNK1 expression, respectively. Promoter analysis identified a potent FLI1 binding site in the regulatory region of the MKNK1 promoter. In transient transfection experiments, FLI1 increased MKNK1 promoter activity, which was blocked by mutating the FLI1 binding site. FLI1 specifically affected the expression of MKNK1, but not that of MKNK2. The siRNA‑mediated downregulation of MKNK1 downregulated the expression of survivin (BIRC5) and significantly suppressed cell proliferation in culture. FLI1 inhibitory compounds were shown to downregulate this oncogene through the suppression of MAPK/extracellular‑regulated kinase (ERK) signaling and the subsequent activation of miR‑145, leading to a lower MKNK1 expression and the suppression of leukemic growth. These results uncover a critical role for FLI1 in the control of protein translation and the importance of targeting its function and downstream mediators, such as MKNK1, for cancer therapy.

The eukaryotic elongation factor-2 kinase, eEF2K, which restricts protein translation elongation, has been identified as a potential therapeutic target for diverse types of malignancies including triple negative breast cancer (TNBC). However, the contexts in which eEF2K inhibition is essential in TNBC and its consequences on the proteome are largely unknown. Here we show that genetic or pharmacological inhibition of eEF2K cooperated with glutamine (Gln) starvation, and synergized with glutaminase (GLS1) inhibitors to suppress growth of diverse TNBC cell lines. eEF2K inhibition also synergized with depletion of eukaryotic translation initiation factor 4E-binding protein 1 (eIF4EBP1; 4EBP1), a suppressor of eukaryotic protein translation initiation factor 4E (eIF4E), to induce c-MYC and Cyclin D1 expression, yet attenuate growth of TNBC cells. Proteomic analysis revealed that whereas eEF2K depletion alone uniquely induced Cyclin Dependent Kinase 1 (CDK1) and 6 (CDK6), combined depletion of eEF2K and 4EBP1 resulted in overlapping effects on the proteome, with the highest impact on the 'Collagen containing extracellular matrix' pathway (e.g. COL1A1), as well as the amino-acid transporter, SLC7A5/LAT1, suggesting a regulatory loop via mTORC1. In addition, combined depletion of eEF2K and 4EBP1 indirectly reduced the levels of IFN-dependent innate immune response-related factors. Thus, eEF2K inhibition triggers cell cycle arrest/death under unfavourable metabolic conditions such as Gln-starvation/GLS1 inhibition or 4EBP1 depletion, uncovering new therapeutic avenues for TNBC and underscoring a pressing need for clinically relevant eEF2K inhibitors.

Wiskott-Aldrich Syndrome, WAS/WAVE, is a rare, X-linked immune-deficiency disease caused by mutations in the WAS gene, which together with its homolog, N-WASP, regulates actin cytoskeleton remodeling and cell motility. WAS patients suffer from microthrombocytopenia, characterized by a diminished number and size of platelets, though the underlying mechanism is not fully understood. Here, we identified FLI1 as a direct transcriptional regulator of WAS and its binding partner WIP. Depletion of either WAS or WIP in human erythroleukemic cells accelerated cell proliferation, suggesting tumor suppressor function of both genes in leukemia. Depletion of WAS/WIP also led to a significant reduction in the percentage of CD41 and CD61 positive cells, which mark committed megakaryocytes. RNAseq analysis revealed common changes in megakaryocytic gene expression following FLI1 or WASP knockdown. However, in contrast to FLI1, WASP depletion did not alter expression of late-stage platelet-inducing genes. N-WASP was not regulated by FLI1, yet its silencing also reduced the percentage of CD41+ and CD61+ megakaryocytes. Moreover, combined knockdown of WASP and N-WASP further suppressed megakaryocyte differentiation, indicating a major cooperation of these related genes in controlling megakaryocytic cell fate. However, unlike WASP/WIP, N-WASP loss suppressed leukemic cell proliferation. WASP, WIP and N-WASP depletion led to induction of FLI1 expression, mediated by GATA1, and this may mitigate the severity of platelet deficiency in WAS patients. Together, these results uncover a crucial role for FLI1 in megakaryocyte differentiation, implicating this transcription factor in regulating microthrombocytopenia associated with Wiskott-Aldrich syndrome.

SMAD1, a central mediator in TGF-β signaling, is involved in a broad range of biological activities including cell growth, apoptosis, development and immune response, and is implicated in diverse type of malignancies. Whether SMAD1 plays an important role in multiple myeloma (MM) pathogenesis and can serve as a therapeutic target are largely unknown.

Lovastatin, an HMG-CoA inhibitor and an effective cholesterol lowering drug, exhibits anti-neoplastic activity towards several types of cancer, although the underlying mechanism is still not fully understood. Herein, we investigated mechanism of growth inhibition of leukemic cells by lovastatin.

Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator induced in many cancers. It is thought to drive tumorigenesis by repressing division, stemness, and/or developmental regulators. Cancers evade immune detection, and diverse immune regulators are perturbed in different tumors. It is unclear how such cell-specific effects are coordinated. Here, we show a profound and cancer-selective role for PRC2 in repressing multiple cytokine pathways. We find that PRC2 represses hundreds of IFNγ stimulated genes (ISGs), cytokines and cytokine receptors. This target repertoire is significantly broadened in cancer vs non-cancer cells, and is distinct in different cancer types. PRC2 is therefore a higher order regulator of the immune program in cancer cells. Inhibiting PRC2 with either RNAi or EZH2 inhibitors activates cytokine/cytokine receptor promoters marked with bivalent H3K27me3/H3K4me3 chromatin, and augments responsiveness to diverse immune signals. PRC2 inhibition rescues immune gene induction even in the absence of SWI/SNF, a tumor suppressor defective in ~20% of human cancers. This novel PRC2 function in tumor cells could profoundly impact the mechanism of action and efficacy of EZH2 inhibitors in cancer treatment.

The retinoblastoma tumor suppressor (pRb) is thought to orchestrate terminal differentiation by inhibiting cell proliferation and apoptosis and stimulating lineage-specific transcription factors. In this study, we show that in the absence of pRb, differentiating primary myoblasts fuse to form short myotubes that never twitch and degenerate via a nonapoptotic mechanism. The shortened myotubes exhibit an impaired mitochondrial network, mitochondrial perinuclear aggregation, autophagic degradation, and reduced adenosine triphosphate production. Bcl-2 and autophagy inhibitors restore mitochondrial function and rescue muscle degeneration, leading to formation of long, twitching myotubes that express normal levels of muscle-specific proteins and stably exit the cell cycle. A hypoxia-induced glycolytic switch also rescues the myogenic defect after either chronic or acute inactivation of Rb in a hypoxia-inducible factor-1 (HIF-1)-dependent manner. These results demonstrate that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program, and they also reveal a novel link between pRb and cell metabolism.

Basal-like breast cancers (BLBC) express a luminal progenitor gene signature. Notch receptor signaling promotes luminal cell fate specification in the mammary gland, while suppressing stem cell self-renewal. Here we show that deletion of Lfng, a sugar transferase that prevents Notch activation by Jagged ligands, enhances stem/progenitor cell proliferation. Mammary-specific deletion of Lfng induces basal-like and claudin-low tumors with accumulation of Notch intracellular domain fragments, increased expression of proliferation-associated Notch targets, amplification of the Met/Caveolin locus, and elevated Met and Igf-1R signaling. Human BL breast tumors, commonly associated with JAGGED expression, elevated MET signaling, and CAVEOLIN accumulation, express low levels of LFNG. Thus, reduced LFNG expression facilitates JAG/NOTCH luminal progenitor signaling and cooperates with MET/CAVEOLIN basal-type signaling to promote BLBC.

Cholesterol plays vital roles in human physiology; abnormal levels have deleterious pathological consequences. In cancer, elevated or reduced expression of cholesterol biosynthesis is associated with good or poor prognosis, but the underlying mechanisms are largely unknown. The limonoid compounds A1542 and A1543 stimulate ERK/MAPK by direct binding, leading to leukemic cell death and suppression of leukemia in mouse models. In this study, we investigated the downstream consequences of these ERK/MAPK agonists in leukemic cells.

CDK4/6 inhibitors are effective against cancer cells expressing the tumor suppressor RB1, but not RB1-deficient cells, posing the challenge of how to target RB1 loss. In triple-negative breast cancer (TNBC), RB1 and PTEN are frequently inactivated together with TP53. We performed kinome/phosphatase inhibitor screens on primary mouse Rb/p53-, Pten/p53-, and human RB1/PTEN/TP53-deficient TNBC cell lines and identified CDC25 phosphatase as a common target. Pharmacological or genetic inhibition of CDC25 suppressed growth of RB1-deficient TNBC cells that are resistant to combined CDK4/6 plus CDK2 inhibition. Minimal cooperation was observed in vitro between CDC25 antagonists and CDK1, CDK2, or CDK4/6 inhibitors, but strong synergy with WEE1 inhibition was apparent. In accordance with increased PI3K signaling following long-term CDC25 inhibition, CDC25 and PI3K inhibitors effectively synergized to suppress TNBC growth both in vitro and in xenotransplantation models. These results provide a rationale for the development of CDC25-based therapies for diverse RB1/PTEN/TP53-deficient and -proficient TNBCs.